Diffusible Signal Factor Signaling Research Articles

Unveiling the Role of Diffusible Signal Factor-Family Quorum Sensing Signals in Regulating Behavior of Xanthomonas and Lysobacter.

Diffusible signal factor (DSF) family signals represent a unique group of quorum sensing (QS) chemicals that modulate a wide range of behaviors for bacteria to adapt to different environments. However, whether DSF-mediated QS signaling acts as a public language to regulate the behavior of biocontrol and pathogenic bacteria remains unknown. In this study, we present groundbreaking evidence demonstrating that RpfFXc1 or RpfFOH11 could be a conserved DSF-family signal synthase in Xanthomonas campestris or Lysobacter enzymogenes. Interestingly, we found that both RpfFOH11 and RpfFXc1 have the ability to synthesize DSF and BDSF signaling molecules. DSF and BDSF positively regulate the biosynthesis of an antifungal factor (heat-stable antifungal factor, HSAF) in L. enzymogenes. Finally, we show that RpfFXc1 and RpfFOH11 have similar functions in regulating HSAF production in L. enzymogenes, as well as the virulence, synthesis of virulence factors, biofilm formation, and extracellular polysaccharide production in X. campestris. These findings reveal a previously uncharacterized mechanism of DSF-mediated regulation in both biocontrol and pathogenic bacteria.

A Stenotrophomonas maltophilia TetR-Like Transcriptional Regulator Involved in Fatty Acid Metabolism Is Controlled by Quorum Sensing Signals.

Stenotrophomonas maltophilia is an environmental bacterium as well as an emerging opportunistic multidrug-resistant pathogen. They use the endogenous diffusible signal factor (DSF) quorum sensing (QS) system to coordinate population behavior and regulate virulence processes but can also respond to exogenous N-acyl-homoserine lactone (AHL) signals produced by neighboring bacteria. The effect of these QS signals on the global gene expression of this species remains, however, unknown. Whole-transcriptome sequencing analyses were performed for exponential cultures of S. maltophilia K279a treated with exogenous DSF or AHLs. Addition of DSF and AHLs signals resulted in changes in expression of at least 2-fold for 28 and 82 genes, respectively. Interestingly, 22 of these genes were found upregulated by both QS signals, 14 of which were shown to also be induced during the stationary phase. Gene functions regulated by all conditions included lipid and amino acid metabolism, stress response and signal transduction, nitrogen and iron metabolism, and adaptation to microoxic conditions. Among the common top upregulated QS core genes, a putative TetR-like regulator (locus tag SMLT2053) was selected for functional characterization. This regulator controls its own β-oxidation operon (Smlt2053-Smlt2051), and it is found to sense long-chain fatty acids (FAs), including the QS signal DSF. Gene knockout experiments reveal that operon Smlt2053-Smlt2051 is involved in biofilm formation. Overall, our findings provide clues on the effect that QS signals have in S. maltophilia QS-related phenotypes and the transition from the exponential to the stationary phase and bacterial fitness under high-density growth. IMPORTANCE The quorum sensing system in Stenotrophomonas maltophilia, in addition to coordinating the bacterial population, controls virulence-associated phenotypes, such as biofilm formation, motility, protease production, and antibiotic resistance mechanisms. Biofilm formation is frequently associated with the persistence and chronic nature of nosocomial infections. In addition, biofilms exhibit high resistance to antibiotics, making treatment of these infections extremely difficult. The importance of studying the metabolic and regulatory systems controlled by quorum sensing autoinducers will make it possible to discover new targets to control pathogenicity mechanisms in S. maltophilia.

The Wsp chemosensory system modulates c-di-GMP-dependent biofilm formation by integrating DSF quorum sensing through the WspR-RpfG complex in Lysobacter

The ubiquitous Wsp (wrinkly spreader phenotype) chemosensory system and DSF (diffusible signal factor) quorum sensing are two important chemically associated signaling systems that mediate bacterial communications between the host and environment. Although these two systems individually control biofilm formation in pathogenic bacteria via the ubiquitous second messenger c-di-GMP, their crosstalk mechanisms remain elusive. Here we present a scenario from the plant-beneficial and antifungal bacterium Lysobacter enzymogenes OH11, where biofilm formation favors the colonization of this bacterium in fungal hyphae. We found that the Wsp system regulated biofilm formation via WspR-mediated c-di-GMP signaling, whereas DSF system did not depend on the enzymatic activity of RpfG to regulate biofilm formation. We further found that WspR, a diguanylate cyclase (DGC) responsible for c-di-GMP synthesis, could directly bind to one of the DSF signaling components, RpfG, an active phosphodiesterase (PDE) responsible for c-di-GMP degradation. Thus, the WspR-RpfG complex represents a previously undiscovered molecular linker connecting the Wsp and DSF systems. Mechanistically, RpfG could function as an adaptor protein to bind and inhibit the DGC activity of unphosphorylated WspR independent of its PDE activity. Phosphorylation of WspR impaired its binding affinity to RpfG and also blocked the ability of RpfG to act as an adaptor protein, which enabled the Wsp system to regulate biofilm formation in a c-di-GMP-dependent manner by dynamically integrating the DSF system. Our findings demonstrated a previously uncharacterized mechanism of crosstalk between Wsp and DSF systems in plant-beneficial and antifungal bacteria.

A Bacterial Isolate Capable of Quenching Both Diffusible Signal Factor- and N-Acylhomoserine Lactone-Family Quorum Sensing Signals Shows Much Enhanced Biocontrol Potencies.

N-Acylhomoserine lactone (AHL) and diffusible signal factor (DSF) molecules are two families of widely conserved quorum sensing (QS) signals. Quorum quenching (QQ) via enzymatic inactivation of QS signals is a promising strategy of biocontrol. In the search for biocontrol agent quenching both AHL and DSF signals, it has been recently identified that DSF-quenching biocontrol agent Pseudomonas sp. HS-18 contains at least three genes (aigA, aigB, and aigC) encoding AHL-acylases displaying strong AHL-acylase activities on various AHLs. Among them, AigA and AigC presented broad-spectrum enzyme activity against AHLs, while AigB preferred longer AHLs. Interestingly, transcriptional expression of aigC could be significantly induced by AHL signals. Heterologous expression of aigA–C in Burkholderia cenocepacia and Pseudomonas aeruginosa resulted in drastically decreased AHL accumulation, virulence factor production, biofilm formation, motility, and virulence on plants. Significantly, the two types of QQ mechanisms in HS-18 showed a strong and much desired synergistic effect for enhanced biocontrol potency against AHL- and DSF-dependent pathogens.

Formin nanoclustering-mediated actin assembly during plant flagellin and DSF signaling

Plants respond to bacterial infection acutely with actin remodeling during plant immune responses. The mechanisms by which bacterial virulence factors (VFs) modulate plant actin polymerization remain enigmatic. Here, we show that plant-type-I formin serves as the molecular sensor for actin remodeling in response to two bacterial VFs: Xanthomonas campestris pv. campestris (Xcc) diffusible signal factor (DSF), and pathogen-associated molecular pattern (PAMP) flagellin in pattern-triggered immunity (PTI). Both VFs regulate actin assembly by tuning the clustering and nucleation activity of formin on the plasma membrane (PM) at the nano-sized scale. By being integrated within the cell-wall-PM-actin cytoskeleton (CW-PM-AC) continuum, the dynamic behavior and function of formins are highly dependent on each scaffold layer's composition within the CW-PM-AC continuum during both DSF and PTI signaling. Our results reveal a central mechanism for rapid actin remodeling during plant-bacteria interactions, in which bacterial signaling molecules fine tune plant formin nanoclustering in a host mechanical-structure-dependent manner.

Whole-Genome Sequencing Analysis of Quorum Quenching Bacterial Strain Acinetobacter lactucae QL-1 Identifies the FadY Enzyme for Degradation of the Diffusible Signal Factor.

The diffusible signal factor (DSF) is a fatty acid signal molecule and is widely conserved in various Gram-negative bacteria. DSF is involved in the regulation of pathogenic virulence in many bacterial pathogens, including Xanthomonas campestris pv. campestris (Xcc). Quorum quenching (QQ) is a potential approach for preventing and controlling DSF-mediated bacterial infections by the degradation of the DSF signal. Acinetobacter lactucae strain QL-1 possesses a superb DSF degradation ability and effectively attenuates Xcc virulence through QQ. However, the QQ mechanisms in strain QL-1 are still unknown. In the present study, whole-genome sequencing and comparative genomics analysis were conducted to identify the molecular mechanisms of QQ in strain QL-1. We found that the fadY gene of QL-1 is an ortholog of Xcc rpfB, a known DSF degradation gene, suggesting that strain QL-1 is capable of inactivating DSF by QQ enzymes. The results of site-directed mutagenesis indicated that fadY is required for strain QL-1 to degrade DSF. The determination of FadY activity in vitro revealed that the fatty acyl-CoA synthetase FadY had remarkable catalytic activity. Furthermore, the expression of fadY in transformed Xcc strain XC1 was investigated and shown to significantly attenuate bacterial pathogenicity on host plants, such as Chinese cabbage and radish. This is the first report demonstrating a DSF degradation enzyme from A. lactucae. Taken together, these findings shed light on the QQ mechanisms of A. lactucae strain QL-1, and provide useful enzymes and related genes for the biocontrol of infectious diseases caused by DSF-dependent bacterial pathogens.

A Quorum Quenching Bacterial Isolate Contains Multiple Substrate-Inducible Genes Conferring Degradation of Diffusible Signal Factor

Quorum quenching, which disrupts quorum sensing (QS) by either degradation of QS signals or interference of signal generation or perception, is a promising strategy for the prevention and control of QS-mediated bacterial infections. Diffusible signal factor (DSF) is widely conserved in many Gram-negative bacterial pathogens. In this study, we developed an efficient method for screening of highly active DSF degradation microorganisms. Among them, Pseudomonas sp. strain HS-18 showed a superior DSF degradation activity. Bioinformatics and genetic analyses showed that at least 4 genes, designated digA to digD, encoding fatty acyl coenzyme A ligase homologues, are responsible for DSF signal degradation. Interestingly, all 4 dig genes were induced by exogenous DSF, with digA being the most significantly induced. Expression of the dig genes in Xanthomonas campestris pv. campestris markedly reduced the accumulation of endogenous DSF, decreased production of virulence factors, and attenuated bacterial virulence on host plants. Similarly, application of strain HS-18 as a biocontrol agent could substantially reduce the disease severity caused by X. campestris pv. campestris These results unveil the molecular basis of a highly efficient DSF degradation bacterial isolate and present useful genes and biocontrol agents for control of the infectious diseases caused by DSF-dependent bacterial pathogens.IMPORTANCE Diffusible signal factor (DSF) represents a family of widely conserved quorum sensing signals involved in the regulation of virulence factor production in many Gram-negative bacterial pathogens. In this study, we developed a novel and efficient method for screening highly active DSF degradation microorganisms. With this method, we identified a bacterial isolate, Pseudomonas sp. strain HS-18, with a superb DSF degradation activity. We further found that strain HS-18 contains 4 genes responsible for DSF signal degradation, and significantly, these were induced by exogenous DSF molecules. These findings unveil the molecular basis of a highly efficient DSF degradation bacterial isolate and present useful methods, genes, and agents for control of the infectious diseases caused by DSF-dependent bacterial pathogens.

Xanthomonas campestris Promotes Diffusible Signal Factor Biosynthesis and Pathogenicity by Utilizing Glucose and Sucrose from Host Plants.

The plant pathogen Xanthomonas campestris pv. campestris produces diffusible signal factor (DSF) quorum sensing (QS) signals to regulate its biological functions and virulence. Our previous study showed that X. campestris pv. campestris utilizes host plant metabolites to enhance the biosynthesis of DSF family signals. However, it is unclear how X. campestris pv. campestris benefits from the metabolic products of the host plant. In this study, we observed that the host plant metabolites not only boosted the production of the DSF family signals but also modulated the expression levels of DSF-regulated genes in X. campestris pv. campestris. Infection with X. campestris pv. campestris induced changes in the expression of many sugar transporter genes in Arabidopsis thaliana. Exogenous addition of sucrose or glucose, which are the major products of photosynthesis in plants, enhanced DSF signal production and X. campestris pv. campestris pathogenicity in the Arabidopsis model. In addition, several sucrose hydrolase-encoding genes in X. campestris pv. campestris and sucrose invertase-encoding genes in the host plant were notably upregulated during the infection process. These enzymes hydrolyzed sucrose to glucose and fructose, and in trans expression of one of these enzymes, CINV1 of A. thaliana or XC_0805 of X. campestris pv. campestris, enhanced DSF signal biosynthesis in X. campestris pv. campestris in the presence of sucrose. Taken together, our findings demonstrate that X. campestris pv. campestris applies multiple strategies to utilize host plant sugars to enhance QS and pathogenicity.

LesR is a novel upstream regulator that controls downstream Clp expression to modulate antibiotic HSAF biosynthesis and cell aggregation in Lysobacter enzymogenes OH11

BackgroundHeat-stable antifungal factor (HSAF) is a polycyclic tetramate macrolactam secondary metabolite that exhibits broad-spectrum inhibitory activities against filamentous fungal pathogens. The native yield of this chemical is low. It is also a great challenge to synthesize HSAF artificially, due to its complex structure. Understanding the regulatory mechanism underlying HSAF biosynthesis could provide genetic basis for engineering high HSAF-producing strain. The transcription factor Clp is a global regulator that controls bacterial pathogenicity and the expression of one hundred related genes in the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc). Diffusible signal factor (DSF) chemical signaling is the only well-characterized upstream regulatory pathway that involves downstream Clp regulation in Xcc. Such a regulatory hierarchy between DSF signaling and Clp is also conserved in the Gram-negative biological control agent Lysobacter enzymogenes, where the DSF signaling system controls antifungal antibiotic HSAF biosynthesis via Clp.ResultsHere, using LLysobacter enzymogenes OH11 as a working organism, we examined a novel upstream regulator, LesR, a LuxR solo that controls Clp expression to modulate HSAF biosynthesis as well as cell aggregation. We found that the overexpression of lesR in strain OH11 almost entirely shut down HSAF production and accelerated cell aggregation. These changed phenotypes could be rescued by the introduction of plasmid-borne clp in the lesR overexpression background. Consistent with findings, we further found that overexpression of lesR led to a decrease in the Clp level.ConclusionsThese results collectively have shown that LesR could exert its function, i.e., HSAF biosynthesis, via downstream Clp. These findings were subsequently validated by a comparative transcriptome analysis, where the regulatory action of LesR was found to largely overlap with that of Clp. Therefore, in addition to the well-known DSF signaling system, the present study reveals that LesR functions as a new upstream regulatory factor of Clp in L. enzymogenes. The key factor was important for the production of HSAF. The strains with high HSAF yield can presumably be constructed by deletion of the negative regulators or overexpression of the positive regulators by genetic engineering.

Glucose 6-phosphate isomerase (Pgi) is required for extracellular polysaccharide biosynthesis, DSF signals production and full virulence of Xanthomonas oryzae pv. oryzicola in rice

Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak (BLS), recruits five carbohydrate catabolic pathways (glycolysis, ED, PPP, gluconeogenesis and TCA cycle) to acquire nutrients from host rice for parasitism and pathopoiesis. Glucose 6-phosphate isomerase (Pgi), which reversibly converts glucose 6-phosphate to fructose 6-phosphate, plays an important role in glycolysis and gluconeogenesis. In this study, we found that Pgi was essential for the pathogenicity of Xoc in rice. The mutagenesis in pgi caused the pathogen unable to effectively utilize fructose, sucrose, mannose and pyruvate for growth, but did not affect bacterial growth when glucose or galactose used as the sole carbon source. The expression of hrpX and hrpG was repressed and the transcription of hrpE, hpa3 and hrcU were enhanced when pgi or clp (encoding cAMP-regulatory protein) was mutated. Intriguingly, the mutation in pgi resulted in the transcription alteration of key genes, rpfF, rpfC, rpfG and clp, in the quorum-sensing (QS) signaling pathway mediated by diffusible signal factor (DSF) signals and significantly reduced DSF signals production compared with the wild-type. Additionally, the pgi deletion not only impaired bacterial growth and virulence in rice, but also reduced bacterial extracellular polysaccharide (EPS) biosynthesis and cell motility. The lost properties mentioned above in RΔpgi were completely restored to the wild-type levels by the presence of pgi in trans. Taken together, these findings exhibited functional roles of Pgi coordinating with the hrp system and the QS system mediated by DSF signals in carbon metabolic pathways.

Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity.

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

Xanthomonas campestris FabH is required for branched-chain fatty acid and DSF-family quorum sensing signal biosynthesis

Xanthomonas campestris pv. campestris (Xcc), a Gram-negative phytopathogenic bacterium, causes black rot disease of cruciferous vegetables. Although Xcc has a complex fatty acid profile comprised of straight-chain fatty acids and branched-chain fatty acids (BCFAs), and encodes a complete set of genes required for fatty acid synthesis, there is still little known about the mechanism of BCFA synthesis. We reported that expression of Xcc fabH restores the growth of Ralstonia solanacearum fabH mutant, and this allows the R. solanacearum fabH mutant to produce BCFAs. Using in vitro assays, we demonstrated that Xcc FabH is able to condense branched-chain acyl-CoAs with malonyl-ACP to initiate BCFA synthesis. Moreover, although the fabH gene is essential for growth of Xcc, it can be replaced with Escherichia coli fabH, and Xcc mutants failed to produce BCFAs. These results suggest that Xcc does not have an obligatory requirement for BCFAs. Furthermore, Xcc mutants lost the ability to produce cis-11-methyl-2-dodecenoic acid, a diffusible signal factor (DSF) required for quorum sensing of Xcc, which confirms that the fatty acid synthetic pathway supplies the intermediates for DSF signal biosynthesis. Our study also showed that replacing Xcc fabH with E. coli fabH affected Xcc pathogenesis in host plants.

Insights into the distinct cooperation between the transcription factor Clp and LeDSF signaling in the regulation of antifungal factors in Lysobacter enzymogenes OH11

The DSF (Diffusible Signal Factor) signaling and transcription factor Clp play key roles in regulating a diverse range of physiology in the phytopathogen Xanthomonas campestris pv. campestris. However, their functional relationship in other bacteria is little addressed. Here, we presented evidences to show that there are distinct differences in signaling connection between the Clp and LeDSF pathways in Lysobacter enzymogenes, which is a Gram-negative bacterium with remarkable antifungal activity. We found that Clp was a bona fide regulator downstream of the LeDSF signaling in modulating HSAF biosynthesis. However, regulations of Clp on the other two processes, namely, the extracellular protease and chitinase productions, as well as twitching motility, were found to be independent of the LeDSF signaling. These distinct behaviors of Clp were further validated by a comprehensive transcriptomic analysis. Taken together, the present study expands our understanding on the connection between the DSF signaling and Clp signaling pathways in different bacteria.

Promiscuous Diffusible Signal Factor Production and Responsiveness of the Xylella fastidiosa Rpf System.

ABSTRACTCell density-dependent regulation of gene expression in Xylella fastidiosa that is crucial to its switching between plant hosts and insect vectors is dependent on RpfF and its production of 2-enoic acids known as diffusible signal factor (DSF). We show that X. fastidiosa produces a particularly large variety of similar, relatively long-chain-length 2-enoic acids that are active in modulating gene expression. Both X. fastidiosa itself and a Pantoea agglomerans surrogate host harboring X. fastidiosa RpfF (XfRpfF) is capable of producing a variety of both saturated and unsaturated free fatty acids. However, only 2-cis unsaturated acids were found to be biologically active in X. fastidiosa. X. fastidiosa produces, and is particularly responsive to, a novel DSF species, 2-cis-hexadecanoic acid that we term XfDSF2. It is also responsive to other, even longer 2-enoic acids to which other taxa such as Xanthomonas campestris are unresponsive. The 2-enoic acids that are produced by X. fastidiosa are strongly affected by the cellular growth environment, with XfDSF2 not detected in culture media in which 2-tetradecenoic acid (XfDSF1) had previously been found. X. fastidiosa is responsive to much lower concentrations of XfDSF2 than XfDSF1. Apparently competitive interactions can occur between various saturated and unsaturated fatty acids that block the function of those agonistic 2-enoic fatty acids. By altering the particular 2-enoic acids produced and the relative balance of free enoic and saturated fatty acids, X. fastidiosa might modulate the extent of DSF-mediated quorum sensing.

The Diffusible Signal Factor Family of Bacterial CellCell Signals

AbstractBacterial cell‐to‐cell signals of the diffusible signal factor (DSF) family are cis‐2‐unsaturated fatty acids that differ in their chain length and branching patterns. Signaling involving DSF family members occurs in diverse bacteria to include plant and human pathogens. In the majority of these organisms, the perception of DSF is linked to turnover of the second messenger cyclic di‐GMP by one of two “core” pathways. Additional “accessory” signal transduction pathways can also be found, but are not widely conserved. DSF signaling acts to regulate diverse functions to include biofilm formation and architecture, antibiotic resistance, and the production of virulence factors in pathogens. DSF family signals can also participate in interspecies signaling with other bacteria and interkingdom signaling with the yeast Candida albicans. Such interactions may have importance in modifying microbial behavior during polymicrobial infections.

The RpfB-Dependent Quorum Sensing Signal Turnover System Is Required for Adaptation and Virulence in Rice Bacterial Blight Pathogen Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, produces diffusible signal factor (DSF) family quorum sensing signals to regulate virulence. The biosynthesis and perception of DSF family signals require components of the rpf (regulation of pathogenicity factors) cluster. In this study, we report that RpfB plays an essential role in DSF family signal turnover in X. oryzae pv. oryzae PXO99A. The production of DSF family signals was boosted by deletion of the rpfB gene and was abolished by its overexpression. The RpfC/RpfG-mediated DSF signaling system negatively regulates rpfB expression via the global transcription regulator Clp, whose activity is reversible in the presence of cyclic diguanylate monophosphate. These findings indicate that the DSF family signal turnover system in PXO99A is generally consistent with that in Xanthomonas campestris pv. campestris. Moreover, this study has revealed several specific roles of RpfB in PXO99A. First, the rpfB deletion mutant produced high levels of DSF family signals but reduced extracellular polysaccharide production, extracellular amylase activity, and attenuated pathogenicity. Second, the rpfB/rpfC double-deletion mutant was partially deficient in xanthomonadin production. Taken together, the RpfB-dependent DSF family signal turnover system is a conserved and naturally presenting signal turnover system in Xanthomonas spp., which plays unique roles in X. oryzae pv. oryzae adaptation and pathogenesis.

The DSF type quorum sensing signalling system RpfF/R regulates diverse phenotypes in the opportunistic pathogen Cronobacter.

Several bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals to control biofilm formation and virulence. Previous work showed that in Burkholderia cenocepacia the RpfFBc/RpfR system is involved in sensing and responding to DSF signals and that this signal/sensor gene pair is highly conserved in several bacterial species including Cronobacter spp. Here we show that C. turicensis LMG 23827T possesses a functional RpfF/R system that is involved in the regulation of various phenotypes, including colony morphology, biofilm formation and swarming motility. In vivo experiments using the zebrafish embryo model revealed a role of this regulatory system in virulence of this opportunistic pathogen. We provide evidence that the RpfF/R system modulates the intracellular c-di-GMP level of the organism, an effect that may underpin the alteration in phenotype and thus the regulated phenotypes may be a consequence thereof. This first report on an RpfF/R-type QS system of an organism outside the genus Burkholderia revealed that both the underlying molecular mechanisms as well as the regulated functions show a high degree of conservation.

Burkholderia Diffusible Signal Factor Signals to Francisella novicida To Disperse Biofilm and Increase Siderophore Production.

In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within the ecological niche. We analyzed the potential interspecies signaling effect of the Burkholderia diffusible signal factor (BDSF) on Francisella novicida, a model organism for Francisella tularensis, and demonstrated that BDSF both inhibits the formation and causes the dispersion of Francisella biofilm. Specificity was demonstrated for the cis versus the trans form of BDSF. Using transcriptome sequencing, quantitative reverse transcription-PCR, and activity assays, we found that BDSF altered the expression of many F. novicida genes, including genes involved in biofilm formation, such as chitinases. Using a chitinase inhibitor, the antibiofilm activity of BDSF was also shown to be chitinase dependent. In addition, BDSF caused an increase in RelA expression and increased levels of (p)ppGpp, leading to decreased biofilm production. These results support our observation that exposure of F. novicida to BDSF causes biofilm dispersal. Furthermore, BDSF upregulated the genes involved in iron acquisition (figABCD), increasing siderophore production. Thus, this study provides evidence for a potential role and mechanism of diffusible signal factor (DSF) signaling in the genus Francisella and suggests the possibility of interspecies signaling between Francisella and other bacteria. Overall, this study suggests that in response to the interspecies DSF signal, F. novicida can alter its gene expression and regulate its biofilm formation.

The DSF Family of Cell-Cell Signals: An Expanding Class of Bacterial Virulence Regulators.

Many pathogenic bacteria use cell–cell signaling systems involving the synthesis and perception of diffusible signal molecules to control virulence as a response to cell density or confinement to niches. Bacteria produce signals of diverse structural classes. Signal molecules of the diffusible signal factor (DSF) family are cis-2-unsaturated fatty acids. The paradigm is cis-11-methyl-2-dodecenoic acid from Xanthomonas campestris pv. campestris (Xcc), which controls virulence in this plant pathogen. Although DSF synthesis was thought to be restricted to the xanthomonads, it is now known that structurally related molecules are produced by the unrelated bacteria Burkholderia cenocepacia and Pseudomonas aeruginosa. Furthermore, signaling involving these DSF family members contributes to bacterial virulence, formation of biofilms and antibiotic tolerance in these important human pathogens. Here we review the recent advances in understanding DSF signaling and its regulatory role in different bacteria. These advances include the description of the pathway/mechanism of DSF biosynthesis, identification of novel DSF synthases and new members of the DSF family, the demonstration of a diversity of DSF sensors to include proteins with a Per-Arnt-Sim (PAS) domain and the description of some of the signal transduction mechanisms that impinge on virulence factor expression. In addition, we address the role of DSF family signals in interspecies signaling that modulates the behavior of other microorganisms. Finally, we consider a number of recently reported approaches for the control of bacterial virulence through the modulation of DSF signaling.

Identification and Characterization of Two Novel DSF-Controlled Virulence-Associated Genes Within the nodB-rhgB Locus of Xanthomonas oryzae pv. oryzicola Rs105.

Xanthomonas oryzae pv. oryzicola and X. oryzae pv. oryzae are two pathovars of X. oryzae that cause leaf streak and blight in rice, respectively. These two bacterial pathogens cause different disease symptoms by utilizing different infection sites on rice. Compared with X. oryzae pv. oryzae, the molecular virulence mechanism of X. oryzae pv. oryzicola remains largely unknown. Previously, we identified a unique diffusible signal factor (DSF)-controlled virulence-related gene (hshB) in X. oryzae pv. oryzicola Rs105 located in the nodB-rghB locus, which is absent in X. oryzae pv. oryzae PXO99(A). In the present study, we identified two additional genes within this locus (hshA and hshC) that were unique to X. oryzae pv. oryzicola Rs105 compared with X. oryzae pv. oryzae PXO99(A), and we found that the transcription of these genes was regulated by DSF signaling in X. oryzae pv. oryzicola. The mutation of these genes impaired the virulence of the wild-type Rs105 when using a low inoculation density of X. oryzae pv. oryzicola. In contrast to hshB, the mutation of these genes did not have any visible effect on characterized virulence-related functions, including in vitro growth, extracellular polysaccharide production, extracellular protease activity, and antioxidative ability. However, we found that mutation of hshA or hshC significantly reduced the in planta growth ability and epiphytic survival level of X. oryzae pv. oryzicola cells, which was the probable mechanisms of involvement of these two genes in virulence. Collectively, our studies of X. oryzae pv. oryzicola have identified two novel DSF-controlled virulence-associated genes (hshA and hshC), which will add to our understanding of the regulatory mechanisms of conserved DSF virulence signaling in Xanthomonas species.